Method for simultaneously detecting polymyxin B and polymyxin E

Abstract

The invention relates to a method for simultaneously detecting polymyxin B and polymyxin E. The method comprises the following steps: (1) taking isotope labeled polymyxin B1, B2, E1 and E2 as internal standards, dissolving, and diluting with methanol containing formic acid to prepare a sample releasing agent; and (2) mixing a detection sample with the sample releasing agent, carrying out vortex, standing and centrifugation, taking a supernatant, carrying out sample introduction, and carrying out detection by adopting a liquid chromatography-tandem mass spectrometry method. The method can be used for synchronously detecting the patient sample containing the polymyxin B and the polymyxin E.

Description

Technical Field

[0001] The invention belongs to the technical field of polymyxin detection, and particularly relates to a method for simultaneously detecting polymyxin B and polymyxin E. Background Art

[0002] Polymyxin B and polymyxin E, two types of polypeptide antibiotics extracted from the fermentation broth of Paenibacillus polymyxa, are known for their excellent antibacterial efficacy. Since the 1950s, they have been widely used in clinical treatment, although they were later gradually replaced by other safer antibiotics due to their greater nephrotoxicity. However, in the current context of increasingly severe multidrug-resistant and extensively drug-resistant Gram-negative bacilli infections, polymyxins have once again become a powerful weapon in clinical treatment with their unique antibacterial effects.

[0003] Polymyxin B and polymyxin E have similar antibacterial spectra and are both narrow-spectrum antibiotics. They show significant antibacterial effects against most Gram-negative bacilli, especially against resistant strains such as Pseudomonas aeruginosa and Acinetobacter. Therefore, they play a key role in the treatment of serious infections caused by resistant Gram-negative bacteria, such as bacteremia, endocarditis, pneumonia, and post-burn infections. In addition, polymyxin B and polymyxin E are also widely used in the treatment of urinary tract infections, eye infections, tracheal infections, meningitis, sepsis, burn infections, and skin and mucosal infections. In view of its potential nephrotoxicity, the clinical use of polymyxins must strictly follow the indications and monitor drug concentrations when necessary to ensure the safety and effectiveness of treatment.

[0004] In terms of chemical structure, polymyxin B and polymyxin E are polypeptides composed of multiple amino acids. Their structures are very similar, and they differ only in the 6th amino acid of the peptide ring: polymyxin B is phenylalanine, while polymyxin E is leucine. It is worth noting that polymyxin is not a single compound, but a complex mixture produced by the fermentation process, which contains multiple components. For example, polymyxin B contains components such as B1, B2, B3, B1-I, B4, and B5 in different proportions. Among these components, B1 and B2 have the highest content in polymyxin B and contribute the most to the efficacy and toxicological effects. Polymyxin E also has a similar situation. Therefore, therapeutic drug monitoring (TDM) also focuses on the concentrations of the four components of polymyxin B1, B2, E1, and E2.

[0005] In clinical practice, serum or plasma samples are usually collected for therapeutic drug monitoring (TDM) of polymyxins. Currently, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is the most widely used detection technology. This technology generally uses organic solvent treatment to achieve protein precipitation and release polymyxin, and then uses a C18 chromatographic column to separate the target analyte from impurities. On a triple quadrupole mass spectrometer, multiple reaction monitoring (MRM) mode is used to selectively detect specific ion pairs of polymyxin. This method has a high specificity and can accurately distinguish the various components of polymyxin; at the same time, it can provide accurate and precise quantitative results within a relatively wide linear range, meeting the requirements of clinical therapeutic drug monitoring.

[0006] In the application of liquid chromatography tandem mass spectrometry (LC-MS / MS), the internal standard is an indispensable key factor. The internal standard plays a dual role in the sample analysis process: one is to calibrate the loss in the sample pretreatment stage; the other is to compensate for the fluctuation of ionization efficiency. Therefore, the ideal internal standard should be a substance that is highly similar to the target analyte in physicochemical properties and does not exist in the sample. The most ideal internal standard is an isotope-labeled compound of the target analyte, which not only has exactly the same physicochemical properties as the target analyte, but also does not exist naturally in the sample and can be effectively distinguished in mass spectrometry detection.

[0007] Since isotope-labeled internal standards of polymyxin B and polymyxin E are difficult to obtain, a mutual internal standard strategy is often used in research. That is, when determining polymyxin B, polymyxin E is used as the internal standard, and vice versa. Due to the special physicochemical properties of polymyxin, no other alternative substances with similar physicochemical properties have been found.

[0008] In the traditional diagnosis and treatment process, polymyxin B and polymyxin E are usually used alone, so the use of each other's internal standard strategy will not cause mutual interference. However, with the evolution of clinical treatment strategies, the combined intravenous and topical use of polymyxin B and polymyxin E has gradually become the norm. In the case of such combined medication, the use of each other's internal standard strategy is no longer applicable. In addition, although polymyxin B and polymyxin E are highly similar in structure, there are still subtle differences between them. These differences may lead to differences in physicochemical properties, which in turn affect the stability of the test results, especially in some special samples.

[0009] Considering that non-isotope labeled internal standard strategies were used in the past, it is crucial to study the absolute recovery of polymyxin in depth to ensure that it is as close to 100% as possible. This requires that the sample pretreatment steps must be precise and complex, usually including acidification, precipitation and other operations. This undoubtedly increases the difficulty of clinical testing and may introduce additional uncertainty in test results. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting polymyxin B and polymyxin E, which can synchronously detect patient samples containing both polymyxin B and polymyxin E.

[0011] The present invention provides a method for simultaneously detecting polymyxin B and polymyxin E, comprising the following steps:

[0012] (1) Using isotope-labeled polymyxin B1, B2, E1, and E2 as internal standards, the samples were dissolved and diluted with methanol containing formic acid to prepare sample release agents;

[0013] (2) The test sample is mixed with the above-mentioned sample release agent, vortexed, allowed to stand, and centrifuged, and then the supernatant is sampled and tested by liquid chromatography-tandem mass spectrometry.

[0014] Preferably, the isotope-labeled polymyxins B1, B2, E1, and E2 in step (1) are obtained by replacing the leucine at position 7 of the natural polymyxin with 13 C6, 15 N-labeled isotope leucine replacement.

[0015] Preferably, the content of formic acid in the methanol in step (1) is 0.1-0.5 v / v%.

[0016] Preferably, the volume ratio of the test sample to the sample release agent in step (2) is 1:3-5.

[0017] Preferably, the parameters of the liquid chromatography tandem mass spectrometry in step (2) are as follows: mobile phase A: ultrapure water containing 0.1 v / v% formic acid; mobile phase B: acetonitrile; flow rate: 0.4 ml / min; chromatographic column temperature: 40°C.

[0018] Beneficial Effects

[0019] (1) The present invention uses isotope-labeled polymyxin B1, B2, E1, and E2 as internal standard substances, whose physicochemical properties are exactly the same as those of naturally occurring polymyxin B1, B2, E1, and E2, and can effectively correct the variation of samples in the pretreatment and ionization stages, thereby ensuring the accuracy and precision of the test results.

[0020] (2) The present invention can simultaneously detect patient samples containing both polymyxin B and E.

[0021] (3) In view of the adoption of the isotope-labeled internal standard strategy, the present invention simplifies the sample pretreatment process, and the sample addition can be achieved in only one step, thereby improving the efficiency and convenience of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the calibration curve of polymyxin B1.

[0023] Figure 2 This is the calibration curve of polymyxin B2.

[0024] Figure 3 is the calibration curve of polymyxin E1.

[0025] Figure 4 is the calibration curve of polymyxin E2.

[0026] Figure 5 is the absolute recovery rate of polymyxin in different extraction solutions. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] Example 1

[0029] 1. Preparation of sample release agent

[0030] Isotope-labeled polymyxin B1, B2, E1, and E2 were used as internal standards (isotope-labeled polymyxin B1, B2, E1, and E2 were obtained by replacing the leucine at position 7 of natural polymyxin with 13 C6, 15 N-labeled isotope leucine replacement) were dissolved in water to prepare a stock solution with a concentration of 1 mg / mL. Take appropriate amounts of various polymyxin stock solutions and mix them with methanol containing 0.1 v / v% formic acid to make the internal standard concentration of each polymyxin reach 333 ng / mL.

[0031] 2. Sample Pretreatment

[0032] ① Take 50 μl of sample and add 150 μl of sample release agent containing internal standard;

[0033] ② Vortex for 5 seconds, let stand for 5 minutes, and vortex again for 5 seconds;

[0034] ③ Centrifuge for 5 minutes in a centrifuge at a centrifugal force of more than 12,000 × g and below 10°C;

[0035] ④Take the supernatant for subsequent testing.

[0036] 3. Instrument conditions

[0037] Mobile phase A: ultrapure water containing 0.1% formic acid;

[0038] Mobile phase B: acetonitrile;

[0039] Mobile phase ratio: elution gradient see the table below

[0040] time Mobile phase B ratio Starting 5 1min 100 1.4min 100 1.5min 5 3.5min 5

[0041] Flow rate: 0.4ml / min;

[0042] Column temperature: 40°C.

[0043] Since the ionization results of polymyxin in the mass spectrometer show a multi-charge distribution, the parent ions with the highest response on different instruments are different, so the ion channel should be determined after comparing the response value and cross-interference. The following are the parent ion and daughter ion parameters in three common ionization modes.

[0044] [M+4H]MS multiple reaction monitoring parameters

[0045]

[0046] [M+3H]MS multiple reaction monitoring parameters

[0047]

[0048]

[0049] [M+2H]MS multiple reaction monitoring parameters

[0050]

[0051] 4. Linear range

[0052] In this study, polymyxin B and E standard substances were prepared into stock solutions and then added into serum in different proportions to reach concentrations of 0.12, 0.6, 3, 6, 12, and 24 μg / mL. The concentrations of polymyxin B1, B2, E1, and E2 were calculated according to the instructions for use of the standard substances.

[0053] The spiked samples were pre-treated according to the method described in the present invention and then injected for testing. A calibration curve was established with the peak area ratio of the standard substance to the internal standard as the ordinate and the concentration as the abscissa. The results are shown in Figure 1-Figure 4 The results showed that the method had good linearity in the range of 0.12-24 μg / mL (correlation coefficient R 2 >0.99).

[0054] 5. Recovery rate

[0055] In this study, three different concentrations of polymyxin B and E spiked samples were prepared for testing, divided into three batches for testing, with 7 parallels each time. The recovery rates are shown in the table below, all between 100-105%.

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[0060]

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[0062]

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[0065]

[0066] 6. Precision

[0067] In this study, three different concentrations of polymyxin B and E spiked samples were prepared for testing, divided into three batches for testing, and seven parallels were tested each time. The spiked concentrations and measured results are shown in the following table. The calculated coefficient of variation of the test results was less than 8.90%.

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[0069]

[0070]

[0071]

[0072]

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[0075]

[0076] Example 2

[0077] Comparison of recovery rates of different extraction reagents

[0078] In this study, spiked samples of polymyxin B and E were prepared and pretreated with different extraction reagents. The suitability of methanol and acetonitrile as protein precipitants was compared, and the suitability of formic acid, trichloroacetic acid (TCA) and ammonium acetate as additives was also verified. In this study, the absolute recovery of polymyxin was calculated, that is, the internal standard was added to the supernatant after sample extraction and separation, which was only used to correct the ionization efficiency, and no correction was made for extraction loss.

[0079] The results are as follows Figure 5 It shows that methanol containing 0.1% formic acid (ie, the extracting solution formula of the present invention) has the highest recovery rate, and is close to 100% absolute recovery rate, while other extracting agents have different degrees of recovery rate loss.

[0080] Example 3

[0081] Comparison of matrix effects using isotopic internal standards and mutual internal standards

[0082] In this study, blank plasma was collected from 6 different individuals and treated with a sample release agent without internal standard. The supernatant was added with polymyxin B, polymyxin E, and isotope-labeled polymyxins B1 and B2 for mass spectrometry detection. The matrix factor and normalized matrix factor were calculated using two strategies: using isotope as internal standard for polymyxin B and using polymyxin E as internal standard for polymyxin B.

[0083] The results showed that matrix 3 would cause an abnormal increase in the response value of polymyxin B (matrix gain). Isotope-labeled polymyxins B1 and B2 can effectively correct the matrix gain. When polymyxin E is used as an internal standard, the amplitude of the matrix gain is not synchronized, which will lead to abnormal test results.

[0084]

[0085]

Claims

1. A method for simultaneously detecting polymyxin B and polymyxin E, comprising the following steps: (1) Using isotope-labeled polymyxin B1, B2, E1, and E2 as internal standards, the samples were dissolved and diluted with methanol containing formic acid to prepare sample release agents; (2) The test sample is mixed with the above-mentioned sample release agent, vortexed, allowed to stand, and centrifuged, and then the supernatant is sampled and tested by liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that: The isotope-labeled polymyxins B1, B2, E1, and E2 in step (1) are obtained by replacing the leucine at position 7 of the natural polymyxin with 13 C6, 15 N-labeled isotope leucine replacement.

3. The method according to claim 1, characterized in that: The content of formic acid in the methanol in step (1) is 0.1-0.5 v / v%.

4. The method according to claim 1, characterized in that: The volume ratio of the test sample to the sample release agent in step (2) is 1:3-5.

5. The method according to claim 1, characterized in that: The parameters of the liquid chromatography tandem mass spectrometry in step (2) are as follows: mobile phase A: ultrapure water containing 0.1 v / v% formic acid; mobile phase B: acetonitrile; flow rate: 0.4 ml / min; chromatographic column temperature: 40°C.

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